Stem cells have the potential to develop into many different cell types in the body during early life and growth. Stem cells can be divided into two broad categories: embryonic and adult. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues. Adult stem cells can differentiate into multiple pathways. Mesenchymal stem cells are adult stem cells which give rise to a variety of cell types: bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendons. Neural stem cells are adult stem cells in the brain which give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal cells—astrocytes and oligodendrocytes. Epithelial stem cells are adult stem cells in the lining of the digestive tract occur in deep crypts and give rise to several cell types: absorptive cells, goblet cells, paneth cells, and enteroendocrine cells. Skin stem cells are adult stem cells which occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells are adult stem cells which give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis.
Hematopoietic stem cells (HSCs) are rare adult stem cells that have been identified in fetal bone marrow, fetal liver, umbilical cord blood, adult bone marrow, and peripheral blood, which are capable of differentiating into three cell lineages including myeloerythroid (red blood cells, granulocytes, monocytes), megakaryocyte (platelets) and lymphoid (T-cells, B-cells, and natural killer) cells. These HSCs are used in clinical transplantation protocols to treat a variety of diseases including malignant and non-malignant disorders. Expansion of HSCs has important clinical applications since the relative inability to expand hematopoietic stem cells ex vivo imposes major limitations on the current use of HSC transplantation. There is shortage of HSCs used for patient treatments related to bone marrow transplantation or genetic disorders. For allogenic bone marrow transplantation, only one third of all patients who would potentially benefit from an HSC transplant will find a suitable human leukocyte antigen (HLA)-matched related donor.
This is especially true in cases where the number of available stem cells is limiting. This includes cord blood-derived stem cells for transplantation into adults and infusion of multiple cord blood units. While these procedures are possibly effective in increasing the overall incidence of engraftment they have not overcome the problem of the slow pace of hematopoietic recovery. Delayed myeloid engraftment after umbilical cord blood transplantation (UCBT) is often associated with increased early transplant related morbidity and mortality. This remains the primary obstacle for the successful use of cord blood as an alternative source of stem cells for allogeneic transplantation and novel strategies are required to overcome this problem.
Bone marrow stem cells have been used to treat a variety of diseases: leukaemia, inflammation, immunology, inborn anomalies of the blood and immune system, aplastic anaemia, and haemoglobinopathies. However, it is difficult and time-consuming to find a matching donor. Only one in three patients will find a suitable donor and many patients die due to being unable to find a proper donor. In addition, finding a proper match is especially problematic for African-Americans, Hispanics, Native Americans and people of mixed ethnicity. Therefore, it is demanding to develop a process for growing hematopoetic stem cells, which may eliminate the need for human donors. Creating a cell bank containing different haplotype of marrow stem cells might enable cells from one donor to generate enough supply for more than 1,000 recipients.
Stem cells may also hold the key to the fight against HIV. Possible methods of manipulating blood cells to make them resistant to HIV infection, includes genetically altering receptors on stem cells that differentiate to T cells. The modified stem cells can then be expanded and introduced to patients with HIV.
At present the standard sources of HSCs are bone marrow and peripheral blood. To obtain marrow cells, donors must undergo multiple aspirations to collect several thousand milliliters of bone marrow, a procedure that is carried out under general anaesthesia. To collect HSCs from the peripheral blood, the donor must be treated with granulocyte colony-stimulating factor to increase the number of circulating HSCs. Both of these procedures entail some risk and significant cost.
An important newer source of HSCs is umbilical cord blood (UCB). Umbilical Cord blood has major advantages over other sources of HSCs, such as from bone marrow and mobilized peripheral blood. Not only is UCB readily available from many of the nearly 50 UCB banks across the U.S., it also shows increased tolerance for mismatches with the host major histocompatability complex (MHC).
In addition to relatively widespread availability, these HSCs have several useful properties, including their decreased ability to induce immunological reactivity. In many cases, use of UCB incurs significantly less graft-versus-host disease compared to other sources of HSCs.
Yet, while there are clear advantages associated with the use of UCB, there are key issues that constitute a critical barrier to expanded use of this source of hematopoietic stem cells. An obstacle to the successful use of umbilical cord blood as a source of stem cells for allogenic transplantation is delayed myeloid engraftment. This results in increased early transplant related morbidity and mortality following umbilical cord blood transfusion. Despite intensive and expensive supportive care, there is still >50% treatment-related mortality during the first 100 days post-transplant due to delayed immune system and platelet recovery which leaves patients vulnerable to opportunistic infections. Infusions of multiple cord blood units have been used as a possible approach to increase overall engraftment, but to date have not solved the problem of slow hematopoetic recovery.
Another barrier to expanded use of UBC is limited HSC numbers per cord at harvest. As cell dose has been shown to be a major determinant of engraftment and survival after UCB transplantation, low stem cell numbers represents the most significant barrier to successful UCB stem cell transplantation.
The ability to expand ex vivo, prior to transplantation, the stem cell components of a single cord blood unit will greatly increase the viability of this treatment modality. Infusing patients with larger numbers of stem cells as opposed the limited cells available in an unexpanded cord blood unit, should greatly increase the likelihood of successful engraftment.
The expansion of non-hematopoietic adult stem cells, including stem cells isolated from organs such as brain, heart, liver, pancreas, kidney, lung, etc., has important clinical applications, particularly as an external source of cells for replenishing missing or damaged cells of tissues or organs.
Moreover, stem cell gene therapy for hematologic genetic disorders is constrained by the inefficiency of gene transfer into early hematopoietic progenitors and stem cells. The barrier that needs to be overcome is to expand the population of genetically modified cells so that sufficient modified cells can be obtained before applied to humans. For instance, children with severe sickle cell disease can be cured with bone marrow transplants. In the case of sick cell disease, one does not need to completely destroy the recipient bone marrow but merely to replace it with enough healthy or genetically corrected stem cells so as to produce sufficient quantities of healthy red blood cells.
Expansion of hematopoietic stem cells (HSCs) has remained an important goal to develop advanced cell therapies for bone marrow transplantation and many blood disorders. During the last two decades, since the first hematopoietic growth factors were identified, there have been numerous attempts to expand HSCs in vitro using purified growth factors that are known to regulate HSCs. However, these attempts have met with limited success. For example, the hematopoietic growth factors fetal liver tyrosine kinase (Flt3) ligand, stem cell factor, and interleukins 6 and 11 promoted self-renewal of murine hematopoietic stem cells. However, only a limited expansion of hematopoietic stem cells compared with fresh input cells was observed (1-3).
Although a number of pluripotent embryonic stem (ES) cell genes are identified, none have emerged as a robust factor for HSC expansion. They exhibit either a limited or no role in expansion of HSCs as reported in the literature. The best studies of pluripotent genes reported to date are OCT4 and Nanog and both are unable to induce expansion of HSCs. This conclusion is also supported by our studies that there is no significant effect on HSC expansion in the tissue culture with forced expression of these genes using a viral vector.
Activation of Notch-1 in cell intrinsic pathways has been studied as a possible means to increase expansion of HSCs, and the studies have shown that the activation of these pathways is able to maintain HSCs with lympho-myeloid repopulation potential. Overexpression of HOXB4 is the most effective method for stem cell expansion reported to date. Recently, Antonchuk et al. showed that retroviral overexpression of HOXB4 for 10 to 14 days in vitro could increase the number of repopulating HSCs by 40-fold compared with fresh bone marrow stem cells (4).
However, even 40-fold increase in repopulating HSCs is not sufficient for a variety of purposes.